Bottom Line:
To confirm the results of the expression profiles, in vitro analysis was performed.The results of the present study suggest that activation of the JAK-STAT and NF-κB pathways was characteristic of EBV(+)DLBCL-E, which may reflect the nature of EBV-positive tumor cells.Targeting these pathways as therapies might improve clinical outcomes of EBV(+)DLBCL-E.

fig03: In vitro assay of Epstein–Barr virus (EBV)-infected B-cell lines. A recombinant EBV expressing enhanced green fluorescent protein (EGFP) and G418 (Neomycin) resistant genes was infected into B-cell lines.20,46 (a) Representative sample of an EBV-infected B-cell line (OCI-Ly7). More than 90% of the cells were successfully infected with EBV. EGFP, enhanced green fluorescent protein; PI, propidium iodide. (b) Latent membrane protein 1 (LMP1) expression after EBV infection. Western blot analysis revealed LMP1 expression after EBV infection into SUDHL6. The protein expression level of LMP1 in OCI-Ly7 was quite low (arrow). Lymphoblastoid cell line (LCL) (EBV-infected transformed B-cell line, UH3) was used as a positive control and actin served as the internal control. (c, d) Gene Set Enrichment Analysis (GSEA) revealed that (c) Signal transducer and activator of transcription 3 (STAT3)23 and (d) nuclear factor kappa B (NF-κB)29 gene sets were enriched in EBV-infected B-cell lines compared with the same cell lines without EBV infection. A detailed description of GSEA enrichment score curves is shown in the legend of Figure2. BCL, B-cell lymphoma; NES, normalized enrichment score. (e) STAT3 activation in germinal center B-cell (GCB)-like diffuse large B-cell lymphoma (DLBCL) cell lines. Western blot analysis showed that STAT3 and phosphorylated STAT3 (pSTAT3) (Tyr705) expression increased after EBV infection into SUDHL6, while expression of pSTAT3 was not detected in OCI-Ly7. A stable STAT3 expression was observed before and after EBV infection in OCI-Ly7. Actin served as the internal control. Each band was converted to a numerical value using Image J software (http://rsbweb.nih.gov/ij/) and the calculated value is shown below each band. (f) EBV infection promotes the activation of NF-κB. EBV-infected GCB-like DLBCL cell lines (SUDHL6 and OCI-Ly7) and one EBV-infected activated B-cell-like DLBCL cell line (OCI-Ly3) were successfully established.20,46 The upper panel shows evaluation of the activation of NF-κB transcription factors by electrophoretic mobility shift assay. The mobility of NF-κB is indicated by the arrow. Each band was converted to a numerical value using Image J. Each EBV-infected cell line shows increased NF-κB activity when compared with that without EBV infection. The lower panel shows the expression level of Lamin-B1, a nuclear protein, in each nuclear extract. Only subtle differences are observed in Lamin-B1 expression between cell lines with EBV infection and those without infection.

Mentions:
The results of the expression profile suggest that STAT3 and NF-κB activation were characteristics of EBV(+)DLBCL-E. However, it is not clear whether the activation reflected the nature of the tumor cells or the non-tumor cell of the samples, and if STAT3 and NF-κB activation was associated with EBV infection into tumor cells. To investigate the association between EBV-infected B-cell lymphoma cells and molecular pathways, we then conducted in vitro functional analysis. A recombinant EBV harboring EGFP and neomycin resistance genes20 was used to infect five DLBCL cell lines, of which two EBV-infected GCB-like DLBCL cell lines (SUDHL6 and OCI-Ly7) and one EBV-infected ABC-like DLBCL cell line (OCI-Ly3) were successfully established showing ≥90% EGFP positivity (Fig.3a). The protein expression of LMP1 in SUDHL6 and OCI-Ly3 was confirmed; however, the expression level in OCI-Ly7 was quite low (Figs3b, S1). SUDHL6 and OCI-Ly7 cell lines were successfully used in studying the protein expression of EBNA2. Expression of EBNA2 was not detected in both cell lines (Fig. S2). Gene expression profiling of EBV-infected cell lines was compared with cell lines before EBV infection using STAT3- and NF-κB-related gene sets (Table S2). Figure3(c,d) shows that the gene sets of STAT3 and NF-κB were enriched in EBV-infected B-cell lines. To confirm the protein expression level, we performed western blot analysis using the cell lines and found elevation of STAT3 and enhanced pSTAT3 expression after EBV infection into SUDHL6, whereas the expression was not observed in OCI-Ly7 (Fig.3e). The expression level of pSTAT3 of EBV-infected OCI-Ly3 was the same as that of EBV-naïve OCI-Ly3 (Fig. S1).

fig03: In vitro assay of Epstein–Barr virus (EBV)-infected B-cell lines. A recombinant EBV expressing enhanced green fluorescent protein (EGFP) and G418 (Neomycin) resistant genes was infected into B-cell lines.20,46 (a) Representative sample of an EBV-infected B-cell line (OCI-Ly7). More than 90% of the cells were successfully infected with EBV. EGFP, enhanced green fluorescent protein; PI, propidium iodide. (b) Latent membrane protein 1 (LMP1) expression after EBV infection. Western blot analysis revealed LMP1 expression after EBV infection into SUDHL6. The protein expression level of LMP1 in OCI-Ly7 was quite low (arrow). Lymphoblastoid cell line (LCL) (EBV-infected transformed B-cell line, UH3) was used as a positive control and actin served as the internal control. (c, d) Gene Set Enrichment Analysis (GSEA) revealed that (c) Signal transducer and activator of transcription 3 (STAT3)23 and (d) nuclear factor kappa B (NF-κB)29 gene sets were enriched in EBV-infected B-cell lines compared with the same cell lines without EBV infection. A detailed description of GSEA enrichment score curves is shown in the legend of Figure2. BCL, B-cell lymphoma; NES, normalized enrichment score. (e) STAT3 activation in germinal center B-cell (GCB)-like diffuse large B-cell lymphoma (DLBCL) cell lines. Western blot analysis showed that STAT3 and phosphorylated STAT3 (pSTAT3) (Tyr705) expression increased after EBV infection into SUDHL6, while expression of pSTAT3 was not detected in OCI-Ly7. A stable STAT3 expression was observed before and after EBV infection in OCI-Ly7. Actin served as the internal control. Each band was converted to a numerical value using Image J software (http://rsbweb.nih.gov/ij/) and the calculated value is shown below each band. (f) EBV infection promotes the activation of NF-κB. EBV-infected GCB-like DLBCL cell lines (SUDHL6 and OCI-Ly7) and one EBV-infected activated B-cell-like DLBCL cell line (OCI-Ly3) were successfully established.20,46 The upper panel shows evaluation of the activation of NF-κB transcription factors by electrophoretic mobility shift assay. The mobility of NF-κB is indicated by the arrow. Each band was converted to a numerical value using Image J. Each EBV-infected cell line shows increased NF-κB activity when compared with that without EBV infection. The lower panel shows the expression level of Lamin-B1, a nuclear protein, in each nuclear extract. Only subtle differences are observed in Lamin-B1 expression between cell lines with EBV infection and those without infection.

Mentions:
The results of the expression profile suggest that STAT3 and NF-κB activation were characteristics of EBV(+)DLBCL-E. However, it is not clear whether the activation reflected the nature of the tumor cells or the non-tumor cell of the samples, and if STAT3 and NF-κB activation was associated with EBV infection into tumor cells. To investigate the association between EBV-infected B-cell lymphoma cells and molecular pathways, we then conducted in vitro functional analysis. A recombinant EBV harboring EGFP and neomycin resistance genes20 was used to infect five DLBCL cell lines, of which two EBV-infected GCB-like DLBCL cell lines (SUDHL6 and OCI-Ly7) and one EBV-infected ABC-like DLBCL cell line (OCI-Ly3) were successfully established showing ≥90% EGFP positivity (Fig.3a). The protein expression of LMP1 in SUDHL6 and OCI-Ly3 was confirmed; however, the expression level in OCI-Ly7 was quite low (Figs3b, S1). SUDHL6 and OCI-Ly7 cell lines were successfully used in studying the protein expression of EBNA2. Expression of EBNA2 was not detected in both cell lines (Fig. S2). Gene expression profiling of EBV-infected cell lines was compared with cell lines before EBV infection using STAT3- and NF-κB-related gene sets (Table S2). Figure3(c,d) shows that the gene sets of STAT3 and NF-κB were enriched in EBV-infected B-cell lines. To confirm the protein expression level, we performed western blot analysis using the cell lines and found elevation of STAT3 and enhanced pSTAT3 expression after EBV infection into SUDHL6, whereas the expression was not observed in OCI-Ly7 (Fig.3e). The expression level of pSTAT3 of EBV-infected OCI-Ly3 was the same as that of EBV-naïve OCI-Ly3 (Fig. S1).

Bottom Line:
To confirm the results of the expression profiles, in vitro analysis was performed.The results of the present study suggest that activation of the JAK-STAT and NF-κB pathways was characteristic of EBV(+)DLBCL-E, which may reflect the nature of EBV-positive tumor cells.Targeting these pathways as therapies might improve clinical outcomes of EBV(+)DLBCL-E.